Autoimmune diseases are diseases arising from aberrant response of the immune system against one's own substances and tissues. There are more than 80 different types of autoimmune diseases that, collectively, amount to the number two cause of chronic illness, and one of the top 10 leading causes of death in women of all age groups up to 64 years.
Significant medical research efforts have been devoted to understanding the mechanism of autoimmune diseases and finding effective diagnosis and treatments therefore. Many autoimmune diseases are now characterized by the presence and undesirable activities of autoantibodies. These autoantibodies recognize and bind to often normal and healthy self antigens, thereby causing significant damages and failures of relevant tissues and organs.
Acquired aplastic anemia, also known as aplastic anemia (AA), is a rare but deadly hematologic disease, characterized by a reduced or abolished production of blood cells by bone marrow. The bone marrow's failure to replenish blood cells is believed to result from the destruction of hematopoietic cells—multipotent stem cells that normally generate all three types of blood cells—red blood cells, white blood cells and platelets. Consequently, patients with AA develop severe symptoms if failed early diagnosis and can be fatal if left untreated. Anemia, a reduction in the number of red blood cells, leads to hemoglobin deficiency and hypoxia (lack of oxygen); leucopenia, a reduction in the number of white blood cells, makes individuals more susceptible to infection; and thrombocytopenia, a reduction in the number of platelets, causes the blood not to clot as easily, leading to increased risk of hemorrhage, bruising and general weakness.
Aplastic anemia can be caused by many intrinsic and environmental factors, such as genetic deficiencies, exposure to toxic chemicals, chemotherapy and other drugs, radiation, viruses and even pregnancy. Those caused by external factors, i.e., acquired aplastic anemia, are more common. One important pathophysiological mechanism of AA is thought to be associated with autoimmune responses, where the body's immune system is falsely elicited to attack and destroy hematopoietic cells in bone marrow. Young et al., Blood, 108:2509-19 (2006). In recent years, immunosuppression has become one of the main AA treatments, along with stem-cell transplantation.
Many autoimmune antigens have been identified by immunoassays with sera from patients with autoimmune diseases. One of such target antigens is moesin—membrane-organizing extension spike protein, found to be reactive to autoantibodies in patients with rheumatoid arthritis (RA). Wagatsuma et al., Mol. Immuol., 33:1171-6 (1996). Moesin was initially identified in bovine uterus and characterized as a possible receptor for heparin. Lankes et al., Biochem J. 251:831-42 (1988). Further studies have characterized moesin as a member of the ezrin-radixin-moesin (ERM) protein family. These are proteins that are primarily expressed in cytoplasm, concentrated in actin rich cell-surface structures. They act as structural linkers between the plasma membrane and the actin cytoskeleton, playing roles in the formation of microvilli, cell-cell adhesion, maintenance of cell shape, cell mobility and membrane trafficking. Later studies have revealed that they are also involved in physiological and pathological signal transductions. Louvet-Vallee, Biol. Cell 92:305-16 (2000).
Sequence and structural analysis of the ERM proteins revealed that they share high degrees of inter-species and inter-molecular homologies. The ERM proteins have three domains: an N-terminal domain called FERM domain (band four-point-one, ezrin, radixin, moesin homology domain) because of its homology with the band 4.1 protein, a central helical domain and a C-terminal tail domain. The C-terminal tail domain binds F-actin while the C-terminal tail domain is responsible for binding to adhesion molecules in the plasma membrane. Louvet-Vallee (2000).
Wagatsuma et al (1996) reported detections of anti-ERM autoantibodies in RA patients. Of the 71 patient sera tested, 24 samples (33.8%) reacted with at least one of the recombinant ERM antigens and 10 samples (14%) reacted with recombinant moesin alone. However, the study did not find significant correlation between the presence of anti-ERM antibodies and clinical manifestation, such as disease duration or stage. Moreover, sera from patients with other autoimmune diseases such as Primary Sojgren's Syndrome (PSS) and systemic lupus erythematosus (SLE) did not show any reactivity to the three ERM proteins.
Shcherbina et al. studied the expression pattern and functional properties of ERM proteins in blood cells. Shcherbina et al., FEBS Letters 443:31-6 (1999). Moesin was found to be the predominant ERM protein expressed in different types of blood cells. Cleavage experiments using the protease calpain showed that moesin is resistant to calpain treatments in intact stimulated lymphocytes, whereas ezrin is sensitive to calpain. Such differential sensitivity to calpain implicates different and specialized functions of these ERM proteins in blood cells. In platelets, moesin is the only ERM protein detected, and its expression varies according to platelet activities. In circulating state, moesin is found to be expressed surrounding smooth-surfaced platelets. When platelets are activated, moesins are found to be expressed at the newly formed micorvilli, suggesting its active roles in modulating platelets functions.
Takamatsu et al reported detection of specific antibodies to moesin in the sera of patients with acquired aplastic anemia (AA). Takamatsu et al., Blood 109:2514-20 (2007). Using ELISA, anti-moesin antibodies were shown at high titers in 25 of 67 (37%) AA patients. Further in vitro studies showed that anti-moesin antibodies from AA patients induced inflammatory cytokines such as TNF-α and IFN-γ, implicating its role in the pathophysiology of the disease. Espinoza et al., Intl. Immu. 21:913-23 (2009); Takamatsu et al., J. Immunol. 182:703 (2009).
One of the challenges in clinical management of autoimmune diseases is the accurate and early identification of the diseases in a patient. Since not all patients with AA are immune-mediated, it is critical to identify a reliable marker to distinguish nonimmune-mediated AA from immune-mediated AA. Means for such distinction are useful for selectively treating targeted AA patients with immuno-suppression therapy. Moreover, measuring antibody titers provides effective monitoring of disease stages and treatment progress. The present application described herein provides these tools and other benefits.